Project Summary/Abstract This project proposal is focused on developing an optimal brain-penetrating DNA-nanoparticle formulation for gene therapy purposes in Glioblastoma treatment. Glioblastoma (GBM) is the most common and aggressive primary brain tumor, but currently available therapies have severe side effects and the disease remains uniformly lethal. Gene therapy is a potentially powerful strategy that has shown promise in preclinical studies. However, effective gene therapy has yet to be achieved in humans due in large part to an inability to achieve widespread distribution of gene vectors and yet tumor-selective gene transfer in the brain, which is required for the highly invasive nature of GBM. We have developed synthetic DNA-carrying nanoparticles capable of avoiding trapping within the brain parenchyma due to a combination of small particle size (<<100 nm) and dense PEG coatings. These systems are capable of penetrating throughout the entire striatum of the rat brain when administered by convection enhanced delivery (CED). Our pilot data further suggests that these DNA loaded brain-penetrating nanoparticles (DNA-BPN) provide much more widespread and increased transgene expression in healthy brain parenchyma and brain tumor tissue in vivo following CED compared to gold- standard DNA nanoparticles that do not efficiently penetrate beyond the site of infusion. We will develop and thoroughly test DNA-BPN formulated with multiple promising core polymers and compare their behavior in vitro, ex vivo and in vivo to gold-standard systems and leading virus-based vectors. Since the first-generation DNA-BPN already appear capable of safely transfecting a large part of the rat brain, we plan to investigate other promising polymers that may provide even higher transfection along with the use of a highly tumor- specific promoter to limit therapeutic transgene expression to cancerous cells. Our hypothesis is that this combined approach will allow all tumor cells, including highly invasive tumor cells that cause tumor relapse, to be selectively transfected, thereby minimizing potential side effects by eliminating gene transfer to healthy cells.